BioMed Central
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Retrovirology
Open Access
Research
Activation and detection of HTLV-I Tax-specific CTLs by Epitope
expressing Single-Chain Trimers of MHC Class I in a rat model
Takashi Ohashi*, Mika Nagai, Hiroyuki Okada, Ryo Takayanagi and
Hisatoshi Shida
Address: Division of Molecular Virology, Institute for Genetic Medicine, Hokkaido University, Sapporo, 060-0815, Japan
Email: Takashi Ohashi* - ohashi-t@igm.hokudai.ac.jp; Mika Nagai - purefood@igm.hokudai.ac.jp;
Hiroyuki Okada - hiro1230@igm.hokudai.ac.jp; Ryo Takayanagi - coffea-arabica@igm.hokudai.ac.jp;
Hisatoshi Shida - hshida@igm.hokudai.ac.jp
* Corresponding author
Abstract
Background: Human T cell leukemia virus type I (HTLV-I) causes adult T-cell leukemia (ATL) in
infected individuals after a long incubation period. Immunological studies have suggested that
insufficient host T cell response to HTLV-I is a potential risk factor for ATL. To understand the
relationship between host T cell response and HTLV-I pathogenesis in a rat model system, we have
developed an activation and detection system of HTLV-I Tax-specific cytotoxic T lymphocytes
(CTLs) by Epitope expressing Single-Chain Trimers (SCTs) of MHC Class I.
Results: We have established expression vectors which encode SCTs of rat MHC-I (RT1.Al) with
Tax180-188 peptide. Human cell lines transfected with the established expression vectors were
able to induce IFN-γ and TNF-α production by a Tax180-188-specific CTL line, 4O1/C8. We have
further fused the C-terminus of SCTs to EGFP and established cells expressing SCT-EGFP fusion
protein on the surface. By co-cultivating the cells with 4O1/C8, we have confirmed that the
epitope-specific CTLs acquired SCT-EGFP fusion proteins and that these EGFP-possessed CTLs
were detectable by flow cytometric analysis.
Conclusion: We have generated a SCT of rat MHC-I linked to Tax epitope peptide, which can be
applicable for the induction of Tax-specific CTLs in rat model systems of HTLV-I infection. We have
also established a detection system of Tax-specific CTLs by using cells expressing SCTs fused with
EGFP. These systems will be useful tools in understanding the role of HTLV-I specific CTLs in
HTLV-I pathogenesis.
Background
Human T-cell leukemia virus type I (HTLV-I) is etiologi-
cally linked to adult T-cell leukemia (ATL) [1,2], a chronic
progressive neurological disorder termed HTLV-I-associ-
ated myelopathy/tropical spastic paraparesis (HAM/TSP)
[3,4], and various other human diseases [5-8]. ATL is a
malignant lymphoproliferative disease affecting a sub-
group of middle-aged HTLV-I carriers characterized by the
presence of mature T cell phenotype [9]. HTLV-I genome
contains a unique 3' region, designated as pX, which
encodes the viral transactivator protein, Tax [10]. Because
of its broad transactivation capabilities [11], it is specu-
Published: 8 October 2008
Retrovirology 2008, 5:90 doi:10.1186/1742-4690-5-90
Received: 22 July 2008
Accepted: 8 October 2008
This article is available from: http://www.retrovirology.com/content/5/1/90
© 2008 Ohashi et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Retrovirology 2008, 5:90 http://www.retrovirology.com/content/5/1/90
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lated that Tax plays a central role in HTLV-I associated
immortalization and transformation of T cells, which may
lead to the development of ATL.
Tax is also known as a major target protein recognized by
cytotoxic T lymphocytes (CTL) of HTLV-I carriers [12]. It
has been reported that the levels of HTLV-I-specific CTL
are quite diverse among HTLV-I carriers and that ATL
patients have impaired levels of HTLV-I specific CTLs in
contrast to the high levels of CTL response in HTLV-I car-
riers with HAM/TSP [13-15]. In addition, it has been
known that HTLV-I Tax-specific CTL response was
strongly activated in ATL patients who acquired complete
remission after hematopoietic stem cell transplantation
[16]. Based on these observations, it is speculated that
HTLV-I-specific immune response may contribute to
repressing the growth of HTLV-I infected cells in the
infected individuals and insufficient host T cell response
against HTLV-I may be a risk factor for ATL.
To understand the mechanism of ATL development, it is
very important to dissect the interplay between the virus-
specific CTLs and HTLV-I infected T cells. We have previ-
ously established a rat model of ATL-like disease, which
allows examination of the growth and spread of HTLV-I
infected cells, as well assessment of the effects of immune
T cells on the development of the disease [17,18]. By using
this model system, we also reported the therapeutic effect
of Tax-coding DNA or peptide against the disease [19,20].
For further analyzing the effects of Tax specific CTLs in the
rat model, it is important to develop effective methods to
activate Tax specific CTLs and to detect the virus-specific
CTLs.
It has been reported that single chain trimers (SCTs) of
MHC-I have the potential to efficiently stimulate and
identify antigen specific T cells in both human and mouse
systems [21,22]. In this system, all three components of
MHC-I complexes, such as an antigen peptide, β2-micro-
grobulin (β2m), and MHC-I heavy chain are covalently
attached with flexible linkers. By linking together the three
components into a single chain chimeric protein, a com-
plicated cellular machinery of normal antigen processing
can be bypassed, leading to stable cell surface expression
of MHC-I coupled with an antigenic peptide of interest. In
addition, a new system has been established to identify
virus-specific T cells using the acquisition mechanism of
epitope/MHC complex by CD8 T cells through MHC/TCR
interaction [23].
In this study, to establish an activation system of Tax-spe-
cific CTLs in our rat model system, we have generated a
SCT of rat MHC-I linked to Tax epitope peptide. We have
also established a detection system of Tax-specific CTLs by
using cells expressing SCTs fused with EGFP. These newly
established systems would be useful tools in understand-
ing the role of HTLV-I specific CTLs in HTLV-I pathogene-
sis.
Results
Production and functional capabilities of peptide-
β
2m-
RT1.Al fusion proteins
To establish an activation system of Tax-specific CTLs
using SCTs of rat MHC-I (RT1.Al), we have constructed
expression vectors as illustrated in Figure 1A. Tax180-188
epitope was previously identified as an RT1.Al-restricted
CTL epitope recognized by a Tax-specific CTL line [20]. As
a negative control in this study, we have chosen a putative
RT1.Al-restricted epitope in the envelope of HIV-1 NL4-3
strain, NLEnv371-379, which was determined to have the
same point as the Tax180-188 epitope scored by epitope
prediction data via http://www.syfpeithi.de[24]. Since the
linker length has been reported to influence the immune
detection of SCTs in a mouse system [21], we have pre-
pared SCTs with Tax180-188 or NLEnv 371–379 peptide
linked by different lengths of spacers. We then performed
an in vitro transfection experiment to assess the effects of
SCTs for the activation of Tax-specific CTLs. The 293T cells
were transfected with pEF/RT1Al, pEF/RT1AlSCNLEnv371
S, pEF/RT1AlSCTax180S, or pEF/RT1AlSCTax180L. These
transfected 293T cells were subsequently used to stimulate
an RT1.Al-restricted HTLV-I Tax180-188-specific CTL line,
4O1/C8. As shown in Figure 1B, 293T/RT1AlSCTax180S
and 293T/RT1AlSCTax180L cells were able to induce IFN-
γ secretion by 4O1/C8. Statistical analysis revealed a sig-
nificant increase of IFN-γ production (P = 0.02) in 293T/
RT1AlSCTax180L cells compared with 293T/RT1AlSCTax
180S. In contrast, 293T/RT1Al, 293T/RT1AlSCNLEnv371
S, and nontransfected 293T cells induced little IFN-γ secre-
tion by the Tax-specific CTLs. We have also confirmed the
induction of TNF-α production by these vectors, although
there was no significant difference observed between
293T/RT1AlSCTax180L and 293T/RT1AlSCTax180S cells
(Figure 1D). These results suggested that Tax180-188/
β2m/RT1.Al SCTs were efficiently expressed on the cell sur-
face of the transfected cells and were recognized by the
epitope-specific CTLs.
Establishment of MOLT-4 cells stably expressing SCTs of
RT1.Al
To examine the effects of rat SCTs expressed on human
cells and the influence of linker length on the activation
of CTLs in more detail, we have introduced the expression
vectors into MOLT-4 cells and established the cells stably
expressing SCTs of RT1.Al with the different linker length.
After selection by G418 and cloning, FACS analysis was
performed to determine the expression level of RT1.Al on
MOLT-4 cells. As shown in Figure 2A, equivalent levels of
SCT expression were confirmed on the surface of MOLT-
4/RT1AlSCTax180S and MOLT-4/RT1AlSCTax180L cells,
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Activation of Tax-specific CTLs by 293T cells expressing SCTs with Tax 180–188 epitopeFigure 1
Activation of Tax-specific CTLs by 293T cells expressing SCTs with Tax 180–188 epitope. (A) Diagram of full-
length rat MHC-I (RT1.Al). (B) Diagram of SCTs encoding Tax180-188 or NLEnv371-379 linked to β2m and RT1.Al molecules
with different lengths of linkers. L1, linker 1; TM, transmembrane region; Cyto, cytoplasmic region. (C and D) The 293T cells
were either untreated or transfected with pEF/RT1Al, pEF/RT1AlSCNLEnv371S, pEF/RT1AlSCTax180S, or pEF/
RT1AlSCTax180L. The 293T cells were then incubated with a Tax-specific CD8+ T cell line, 4O1/C8. Production of IFN-γ (C)
and TNF-α (D) in the supernatants was measured by ELISA after 24 hours of culture. The data represent the mean ± the SD of
triplicate wells. Similar results were obtained in two independent experiments.
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Establishment of MOLT-4 cells stably expressing SCTs of RT1.Al
Figure 2
Establishment of MOLT-4 cells stably expressing SCTs of RT1.Al. (A) MOLT-4 cells were transfected with various
SCT expression vectors. After selection by G418 and cloning, flow cytometric analysis was performed to determine the
expression level of RT1.Al on MOLT-4 cells. The percentage of RT1.Al-positive cells is indicated in each part. (B and C) The
MOLT-4 cells expression with indicated SCTs were incubated with a Tax-specific CD8+ T cell line, 4O1/C8. Production of
IFN-γ (B) and TNF-α (C) in the supernatants was then measured by ELISA after 24 hours of culture. The data represent the
mean ± the SD of triplicate wells. Similar results were obtained in two independent experiments.
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whereas we detected higher mean fluorescence intensity
(MFI) in MOLT-4/RT1AlSCNLEnv371S compared with
the other 2 SCT-transfected cells. These SCTs expressing
MOLT-4 cells were subsequently used to stimulate 4O1/
C8 cells. As shown in Figure 2B and 2C, MOLT-4/
RT1AlSCTax180S and MOLT-4/RT1AlSCTax180L cells
were able to induce both IFN-γ and TNF-α secretions by
4O1/C8. MOLT-4/RT1AlSCTax180L induced significantly
higher levels of IFN-γ and TNF-α than those induced by
MOLT-4/RT1AlSCTax180S, suggesting that the SCT with
the longer linker has a higher affinity to the epitope-spe-
cific TCR. In contrast, MOLT-4/RT1AlSCNLEnv371S cells
induced little IFN-γ and TNF-α secretion by the Tax-spe-
cific CTLs, despite the higher expression of SCTs. Parental
MOLT-4 cells did not stimulate the cytokine secretion,
either. These results indicated that the SCTs with longer
linkers have the advantage to efficiently stimulate the
epitope-specific CTLs and suggested that the longer form
would be suitable for further application of immunologi-
cal study.
Inhibitory effects of SCTs expressing Tax180-188 on the
growth of Tax-specific CTLs
We next examined whether the SCTs could induce the
expansion of epitope-specific CTLs in vitro. A series of
SCT-expressing MOLT-4 cell lines were fixed with forma-
lin and then used as stimulators for 4O1/C8. An HTLV-I
infected syngeneic rat cell line, FPM1.BP, was also used as
a stimulator, because it has been used to stimulate 4O1/
C8 cells and was thus known to induce the proliferation
of the CTLs. After 3 days of mixed culture, the growth of
4O1/C8 was evaluated. As shown in Figure 3A, FPM1.BP
cells significantly enhanced the growth of 4O1/C8 as
compared with untransfected MOLT-4 cells. In contrast,
MOLT-4 cells expressing SCTs with Tax180 did not induce
the proliferation of 4O1/C8, but significantly inhibited
the growth of the CTLs. We detected stronger growth inhi-
bition in MOLT-4 cells with longer linkers than those with
shorter linkers. The expression of SCTs with NLEnv371 on
MOLT-4 cells caused no influence on the growth of 4O1/
C8. We also assessed the IFN-γ production in the mixed
culture and confirmed the significantly high level of the
cytokine in the culture of FPM1.BP. It was of note that
IFN-γ production was inversely correlated with the growth
of 4O1/C8 among the mixed cultures of MOLT-4 cells
with different SCTs, suggesting that observed growth inhi-
bition was due to the activation induced cell death
(AICD). Thus, we further investigated the apoptotic status
of 4O1/C8 by Annexin V staining. As shown in Figure 3C,
we observed the increase of Annexin V positive cells after
mixed culture with MOLT-4 cells expressing SCTs with
Tax180, but not with those expressing SCTs with
NLEnv371S. As correlated with the growth inhibition, the
SCTs with longer linker induced higher rate of apoptosis
in 4O1/C8 cells than those with shorter linker did. It is of
note that a much higher level of apoptosis was observed
in the mixed culture of FPM1.BP cells, indicating that
FPM1.BP was able to promote the growth of 4O1/C8 even
though it induced a higher level of AICD at the same time.
To understand the mechanism of enhanced proliferation
induced by FPM1.BP, we have assessed the IL-2 concentra-
tion in the mixed culture and found that production of
the T cell-stimulatory cytokine was dramatically enhanced
by FPM1.BP cells (Figure 3D). These results suggested that
the growth inhibition by SCTs with Tax resulted from
both an enhanced level of AICD and a reduced activation
of proliferation signal(s) including IL-2 pathways, which
FPM1.BP cells were able to stimulate.
Detection of Tax-specific CTLs by SCTs fused with EGFP
To establish a detection system of Tax-specific CTLs, the
single chain peptide-RT1.Al construct was then fused at its
C-terminal end to EGFP as illustrated in Figure 4A. We
have prepared two constructs with covalently linked
Tax180-188 or NLEnv371-379 peptides with longer link-
ers, which were designated as pEF/RT1AlSCTax180L-
EGFP and pEF/RT1AlSCNLEnv371L-EGFP, respectively.
We have also generated a construct, which can express
only RT1.Al fused at its C-terminus to EGFP (pEF/RT1Al-
EGFP). These vectors were transfected into 293T cells to
express fusion proteins on the surface. To determine
whether SCTs with EGFP are properly expressed on the
surface of 293T cells, we have incubated the transfected
293T cells with 4O1/C8 and then assessed the IFN-γ and
TNF-α production in the mixed culture. As shown in Fig-
ure 4B and 4C, neither 4O1/C8 cells mixed with parental
293T nor those with 293T/RT1Al-EGFP produced detecta-
ble levels of IFN-γ and TNF-α. When we pulsed the 293T/
RT1Al-EGFP with 10 μM of Tax180-188 peptides, but not
with NLEnv371-379 peptides, for 30 min before co-culti-
vation, we clearly detected the increase of IFN-γ and TNF-
α production in the culture. The 293T cells expressing
RT1AlSCTax180L-EGFP also induced IFN-γ and TNF-α
production, but those expressing RT1AlSCNLEnv371L-
EGFP did not. These results indicated that RT1.Al-EGFP
fusion proteins with epitope peptides were efficiently rec-
ognized by Tax-specific CTLs.
To determine whether SCTs with EGFP can be acquired by
antigen-specific CTLs, we incubated the transfected 293T
cells together with 4O1/C8 cells or another CD8+ syn-
geneic T cell line, G14, which is not specific to Tax 180-
188. As shown in Figure 5A, more than 60% of 4O1/C8
cells appeared to be positive for EGFP after mixed culture
with 293T/RT1AlSCTax180L-EGFP cells for 1 hour, but
not with 293T/RT1AlSCNLEnv371L-EGFP. In contrast, we
were unable to detect G14 cells acquiring EGFP after
mixed culture with 293T/RT1AlSCTax180L-EGFP. To con-
firm the acquisition of SCT-EGFP fusion proteins by 4O1/
C8, we examined the cells by confocal microscopy. As